Grant-in-Aid for Scientific Research (C)
|Allocation Type||Single-year Grants|
Bacteriology (including Mycology)
|Research Institution||Jichi Medical School|
KIRIKAE Teruo Jichi Medical School, Department of Microbiology, Assistant Professor, 医学部, 講師 (50192563)
|Project Period (FY)
1996 – 1997
Completed(Fiscal Year 1997)
|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1997 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1996 : ¥1,100,000 (Direct Cost : ¥1,100,000)
|Keywords||ENDOTOXIN / LPS / CD14 / ST2 CELLS / IL-6 / モノクロナール抗体 / ST2細胞 / IL-6|
The present study was done to elucidate the biological properties of LPS receptor (s) and its signal transduction system, and to identify some molecules of them. A cell line, ST2, derived from murine bone marrow stroma, was mainly used in the present study. We examined the following points ; a) responsiveness to LPS antagonists and LPS agonists, b) detection of LPS-binding proteins on the ST2 cells and production of polyclonal antisera against these proteins, c) detection of tyrosine-phosphorylated proteins on the ST2 cells. We identified some molecules which is related to LPS-induced activation of ST2 cells.
1) Adaptation of CD14-negative marrow stromal ST2 cells in serum-free medium and LPS-responsiveness of ST2 cells
To exclude completely the effects of CD14 and serum components on LPS responsiveness, we established a subclone of CD14-negative marrow stromal ST2 cells growing under serum-free conditions.
2) Detection of LPS-binding proteins on ST2 sells growing under serum-free conditions.
A ligand-botting method and a cross-linker method using radiolabelled LPS demonstrated a number of LPS-binding proteins on ST2 cells.
3) Production of polyclonal antisera against membrane fraction containing LPS-binding proteins on ST2 cells.
Rabbits were immunized with partial purified LPS-binding proteins on ST2 cells and polyclonal antisera were collected. The biological immunological properties were determined.
4) Detection of tyrosine-phosphorylated proteins on ST2 sells growing under serum-free conditions.
A immunoprecipitation and western blotting methods revealed tyrosine-phosphorylated proteins on ST2 sells stimulated with LPS.
5) Production of monoclonal antibodies against LPS-binding proteins and their associated proteins on ST2 cells.
On based our previous finding, we produced monoclonal antibodies against LPS-binding proteins and their associated proteins on ST2 cells.